Henry Ford Hospital Medical Journal

Volume 6 | Number 2 Article 6

6-1958 Bacterial Susceptibility To Antibiotics Joseph P. Truant

Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons

Recommended Citation Truant, Joseph P. (1958) "Bacterial Susceptibility To Antibiotics," Henry Ford Hospital Medical Bulletin : Vol. 6 : No. 2 , 189-195. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol6/iss2/6

This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. For more information, please contact [email protected]. BACTERIAL SUSCEPTIBILITY TO ANTIBIOTICS

JOSEPH P. TRUANT*

The determination of in vitro susceptibility testing of bacteria to antibiotics has been of considerable value in assisting the clinician to choose the most effective thera­ peutic agent in order to overcome an infectious process. A great deal of valuable time may be lost, often to the detriment of the patient if bacterial cultures and in vitro susceptibility tests are not initiated as soon as possible. The information gained by following this procedure is not only important as an aid in determining which anti­ biotic may be effective against a possible pathogen, but also which antibiotics are in­ effective.

As the scope of antibiotic therapy has been expanded by the discovery and de­ velopment of many new chemotherapeutic agents, the problem of selecting the most suitable one for any individual case has become more and more complex. As a result, the following system has been adopted in the bacteriology laboratory. Routinely, with requests for sensitivity, the pathogenic bacteria are tested in one, two or possibly three stages. If stage one shows the organism to be sensitive to one of the antibiotics tried, stage two will not be undertaken unless there is a specific request by the clinician.

For Gram-positive bacteria, the first stage includes testing with sensitivity discs which contain penicillin, dihydrostreptomycin, tetracycline and chloramphenicol. In the second stage erythromycin, novobiocin and occasionally oleandomycin (matromy- cin) are employed. In reserve are such anti-bacterial agents as bacitracin, furacin, furadantin, neomycin and the sulfas which are employed only after consultation with the clinician.

For the Gram-negative bacteria the first stage includes dihydrostreptomycin, tetracycline and chloramphenicol. Strains of Proteus are tested wflh penicillin and Pseudomonas strains are tested with polymyxin in addition to the mentioned antibiotics during the first trial. In the second stage the clinician may choose from the following; furacin, furadantin, neomycin, polymyxin and the sulfas.

The sensitivity of any given strain or organism to an antimicrobial agent is generally defined in terms of concentration of the agent which inhibits the growth partially or completely. Several methods of determining this value are in common use and the results obtained by the different methods may show some discrepancy, depending on the details of the method and the choice of end point.

In this laboratory, we employ the disc technique since it provides a satisfactory and practical routine procedure for the sensitivity evaluation of microorganisms to a variety of chemotherapeutic agents. The antibiotic discs most commonly employed are listed in Table I. It should be emphasized that this technique is a qualitative pro­ cedure and is designed to determine whether the organisms are sensitive or resistant according to the following scheme;

"Division of Microbiology, Department of Laboratories

189 Bacterial Susceptibility to Antibiotics Sensitive — a marked zone of inhibition around the discs of both high and low series. Moderately sensitive — minimal if any detectable zone of inhibition around the low series disc and a distinct zone of inhibition around the high series disc. Resistant — absence of a zone of inhibition around the discs of both con­ centrations.

TABLE I CONCENTRATIONS OF ANTIBIOTIC DISCS

Low series High Series Penicillin 2 units 10 units Dihydrostreptomycin 10 meg. 50 meg. Tetracycline 5 meg. 30 meg. Chloramphenicol 5 meg. 30 meg. Erythromycin 2 meg. 15 meg. Novobiocin 5 meg. 30 meg. Oleandomycin 2 meg. 15 meg. Neomycin 5 meg. 30 meg. Polymyxin 5 meg. 30 meg. Furacin 100 meg. 100 meg. Furadantin 100 100 n:c-.

During the past year our interest has been centered on methods for controlling the variables inherent in routine disc sensitivity testing. A large number of Gram- positive and Gram-negative isolates from the routine services were tested for sensi­ tivity to several antibiotics by three different methods. In this manner it was possible to reveal the frequency, extent and direction of the variations in the results obtained with the different procedures as applied to antibiotics which are now in general use. It was hoped, in this manner also to elucidate some of the factors responsible for the discrepancies which occur occasionafly.

MATERIALS AND METHODS

Two hundred and thirty bacterial strains and ten antibacterial agents were included in this study. The three methods which were employed for susceptibility testing were as follows; (1) The filter paper "disc agar diffusion" technique (2) The tube or broth dilution technique (3) The cylinder-plate method The details concerning these procedures are clearly discussed by Grove and Randall (1) as well as by Jackson et al (2).

190 Truant In order to obtain the best comparison and evaluation of the different methods, the tube dflution and the cylinder-plate tests done with any given combination of organisms and antibiotics, were set up and carried out using the same seed culture and the same freshly prepared dflutions of antibiotics. From three to six organisms were tested with the antibiotics at one time. The tests employing the medicated discs were carried out separately in our laboratory and some .strains were also tested by the Division of Labora­ tories, Michigan Department of Health. The results were also compared with those reported by the technicians on the routine services.

RESULTS It is not feasible to record the results of all tests with every combination of organisms and antibiotics included in this study. However, sufficient data will be reported in tabular form to illustrate the highlights which were brought out by this investigation.

Table II shows the results of testing 130 staphylococci against penicillin, tetra­ cycline, dihydrostreptomycin. chloramphenicol, oleandomycin, erythromycin and novo­ biocin by the three methods. Inspection of the data shows only small differences in the values obtained by the different procedures. Thus, in almost all instances, agreement was observed. Minor discrepancies (one-dilution differences) occasionally would have altered the reporting in lieu of changing the sensitivity pattern from one category to another. This is practically only important with organisms on the borderline between moderately resistant and resistant, since the latter interpretation would exclude therapy.

The antibiotic spectrum of the staphylococci shown in Table II may be of practical value since it is based on a large number of recent isolates obtained from specimens collected in this hospital. From this spectrum it can be seen that as many as 78.8 per cent of the isolates may be resistant to penicillin. The spectrums of the other anti­ biotics shown in Table II correspond to tho.se reported by other investigators.

Table III shows the results of the susceptibility tests wflh the Gram-negative bacilli. The data shows a greater variation in the sensitivity pattern due to the different methods than the results obtained using similar procedures against the staphylococci. The greatest discrepancy occurred with dihydrostreptomycin using the tube dilution procedure. According to Garrod (4) and others, the levels of resistance to streptomycin with in vitro susceptibility tests may vary with changes in the size of the inoculum. Branch et al (5) have shown that the size of the inoculum is an important factor in changing the minimum inhibitory concentration in the tube dilution method and it also has a slight effect in the agar well method but has not been found to exert any demonstrable changes in the reading of the 'disc results'. Furthermore, as already noted by others (2, 3). additional factors such as media, period of incubation, choice of size of inhibition zone etc. may account for some of the variation in the results shown in Table IIL

The data in Table III shows dihydrostreptomycin and furacin were the most effective antibiotics in vitro against the Gram-negative bacilli. The least effective of the five agents was tetracycline. Previous experience has shown that peniciflin is ineffective against this group of organisms. Occasionafly high concentrations of penicillin will inhibit members of the genus Proteus.

191 TABLE II

COMPARATIVE TESTS BY THREE lETHODS ON 130 STAPHYLOCOCCI

5: Penicillin Tetracycline Dihydro- Chloramphenicol Hatromycin Erythromycin Novobiocin streptomycin METHODS

DISC ^, % sensitive^,j^^ 21.2 42.4 44.3 90.0 97.7 93.4 100 -: % resistant 78.8 57.6 55.7 10.0 2,3 6.6 0 ••3

TUBE DILUTIOI % sensitive 18.2 42.4 44.3 88.6 97.7 90.4 100 % resistant 81.8 57.6 55.7 11.4 2.3 9.6 0 CYLINDER-PLATE % sensitive 22.7 39.4 44.3 87.1 95.5 93.4 100 % resistant 77.3 60.6 55.7 12.9 4.5 6.6 0

* Organisms inhibited ly the range of concentrations listed in Table I for each antibiotic.

** Those species which are not inhibited by the antibiotic concentration shown in Table I. TABIE III

COITARATIVE TESTS BY THREE HETOODS ON 100 GRAM-NEGATIVE BACILLI

Tetracycline Dihydro- Chloramphenicol Furacin Fttradantin streptonycin

METHODS

DISC % sensitive 36.2 80.0 67.5 82.0 54.5 % resistant" 63.8 20.0 32.5 18.0 45.5 TUBE DILUTION % sensitive 42.5 62.3 60.0 75.0 50.0 % resistant 57.5 37.7 40.0 25.0 50.0 CYLBIDHl-PLATE % sensitive 37.5 73.8 60.0 73.0 63.6 % resistant 62.5 26.2 40.0 27.0 36.4

* Organisms inhibited by the range of concentrations listed in Table I for each antibiotic.

Those species which are not inhibited by the antibiotic concentrations shown in Table I. Bacterial Susceptibility to Antibiotics

DISCUSSION Certain conclusions are apparent after an examination of the foregoing data. As might be expected, discrepancies do occur among in vitro results which have been obtained by different methods. None of the procedures lend themselves to accurate quantitation, although reliability and accuracy can be increased by multiple replications, this approach is generally impractical for the routine bacteriological laboratory. It is not meant to imply that tests for susceptibility of bacteria to antibiotics have no value whatever. It is more proper to infer from our experiences and those of other investigators that the tests can be used as a general qualitative guide to distinguish susceptible from non-susceptible organisms. The disc was designed for simplicity and not for a high degree of accuracy. It appears to be adequate for all practical purposes since numerous reports attest to the fact that the results obtained by the disc mehod correlate well with clinical experience. The tube dilution method gives an end point reading and demonstrates the minimum inhibitory concentration (M. I. C.) of the antibiotic. While the information is reliable within the limits of error inherent in such a procedure, the method has serious draw­ backs. These include the large amount of equipment, material, time and effort consumed in testing the sensitivity of an organism to several antibiotics. Therefore, the tube dilution procedure is employed only in situations wherein 'quantitative results' or synergistic studies with two or more drugs are of considerable clinical importance.

The cylinder-plate assays compared favorably with the results of the other two methods. This technique has the disadvantage of being more laborious than the disc method but, like the tube dilution procedure, it has the advantage of being more reliable and accurate. However, the cylinder-plate method is not as yet a practical procedure for routine susceptibility testing although it is extremely useful as an ex­ perimental tool.

Susceptibility studies which have been performed over a period of years with such groups of organisms as Meningococcus, Gonococcus, Pneumococcus, HemophUus and Streptococcus pyogenes (Lancefield Group A) have been more or less uniform in their sensitivity to many antibiotics. The situation is entirely different with a number of other organisms such as the staphylococci, enterococci, coliforms, Proteus, Pseudomonas etc. which vary widely in this respect. For this reason, standard sensi­ tivity tables reported in the literature or distributed by pharmaceutical houses cannot be relied on for the selection of antibiotics most apt to be successful in treating this group of bacteria. SUMMARY The susceptibility patterns of two hundred and thirty organisms to ten antibiotics have been examined by means of three methods. The data shows some degree of variation due to the different testing techniques. The value and limitations of the results of susceptibility tests to antibiotics are discussed, particularly with respect to their clinical application. ACKNOWLEDGMENT I wish to express my appreciation to Alex Dombroski and Alex Mercer for technical services.

194 r Truant

BIBLIOGRAPHY 1. Grove D. C, and Randall, W. A.: Assay Methods of Antibiotics, New York, Medical fcncyclopedia. Inc., 1955. 2. Jackson, G. G., and Finland, M.: Comparison of methods for determining sensitivity of bacteria to antibiotics in vitro, A. M. A. Arch. Int. Med. 88:446, 1951. 3. Greenberg, L., Fitzpatrick, K. M., and Branch, A.: The status of the antibiotic disc in Canada, Canad. M. A. J. 76:194, 1957. 4. Garrod, L. P.: The Bactericidal action of streptomycin, Brit. M. J. 1:382, 1948. 5. Branch, A., Starkey, D. H., Rodgers, K. C, and Power, E. E.: Experience with con­ trolling antibiotic sensitivity tests in department of Veterans Affairs Hospital Laboratories in Canada, Antibiotics Annual, 1954-1955, New York, Medical Encyclopedia, Inc., 1955, p. 1125. 6. Leper, M. H., Moulton, B., Dowling, H. F., Jackson, G. G., and Kofman, S.: Epidemiology of erythromycin-resistant staphylococci in a hospital population; effect on therapeutic activity of erythromycin. Antibiotics Annual, 1953-1954, New York, Medical Encylopedia Inc 1954 p 308

195